Compounds for intracellular delivery of therapeutic moieties to nerve cells

ABSTRACT

A compound for delivering a non-cytotoxic therapeutic moiety into nerve cells, the compound having the general formula:
 
B-L-TM
 
where:
         B is a binding agent capable of selectively binding to a nerve cell surface receptor and mediating absorption of the compound by the nerve cell;   TM is a therapeutic moiety which has a non-cytotoxic therapeutic effect when absorbed by a nerve cell; and   L is a linker coupling B to TM.

FIELD OF THE INVENTION

The present invention relates to compounds which can be used toselectively deliver moieties to nerve cells. More specifically, theinvention relates to compounds which include a therapeutic moiety andfacilitate absorption of the therapeutic moiety by nerve cells.

BACKGROUND OF THE INVENTION

Our understanding of the structure and function of the nervous systemhas been greatly advanced owing to enormous progresses made in field ofneuroscience. Cellular and molecular mechanisms of neuron growth anddevelopment and diseases associated with the central and peripheralnervous systems are studied extensively by using rapidly growingtechniques in molecular and cell biology. However, a need still existsfor efficacious treatments of many neurological disorders includingAlzheimer's disease, Parkinson's disease, Huntington's disease,schizophrenia, severe pain, multiple sclerosis, bipolar disease, anddiseases of the nervous system due to infection by viruses and othermicroorganisms (herpes simplex, HIV, cytomegalovirus, parasites, fungi,prion, etc.).

Many neuropharmaceutical agents have been developed to treat diseases ofthe nervous system, but their usefulness has been hampered by severeside effects partially due to nonspecific interactions between theseagents and cells or tissues other than the targeted cells. For example,steroid hormone cortisone and its derivatives are widely used to treatinflammation in the body including the nerve system to reduce symptomssuch as swelling, tenderness and pain. However, the steroid dosage hasto be kept at the lowest effective level because of its severe sideeffects. Steroid hormone binds to its cognate nuclear hormone receptorand induces a cascade of cellular effects, including programmed celldeath of the neurons in the brain (Kawata M., et al., J. SteroidBiochem. Mol. Biol. 65: 273-280 (1998)). Since steroid hormonereceptors, such as glucocorticord receptor for cortisone, distribute ina wide variety of tissues and cells, nonspecific interactions of thehormone with its cognate receptor in different sites is unavoidable ifthe drug is circulated systemically.

A need continues to exist for an effective system for deliveringtherapeutic agents selectively to nerve cells and nerve tissues. Varioustechniques have been developed to deliver drugs, but with only limitedsuccess. For example, liposomes have been used as carrier molecules todeliver a broad spectrum of agents including small molecules, DNAs,RNAs, and proteins. Liposome mediated delivery of pharmaceutical agentshas major drawbacks because of its lack of target specificity. Attemptshave been made to overcome this problem by covalently attaching wholesite-specific antibody or Fab fragments to liposomes containing apharmaceutical agent (Martin et al., Biochem. 20, 4229-4238, (1981)).However, an intrinsic problem of particular importance in any liposomecarrier system is that in most cases the targeted liposome does notselectively reach its target site in vivo. Whether or not liposomes arecoated with antibody molecules, liposomes are readily phagocytosed bymacrophages and removed from circulation before reaching their targetsites.

SUMMARY OF THE INVENTION

Compounds of the present invention include compounds having the generalformula:B-L-M

where:

-   -   B is a binding agent capable of selectively binding to a nerve        cell surface receptor and mediating absorption of the compound        by the nerve cell;    -   M is a moiety which performs a useful non-cytotoxic function        when absorbed by a nerve cell; and    -   L is a linker coupling B to M.

In one embodiment, the compounds have the general formula:B-L-TM

where:

-   -   B is a binding agent capable of selectively binding to a nerve        cell surface receptor and mediating absorption of the compound        by the nerve cell;    -   TM is a therapeutic moiety which has a non-cytotoxic therapeutic        effect when absorbed by a nerve cell; and    -   L is a linker coupling B to TM.

In another embodiment, the compounds have the general formula:B-L-IM

where:

-   -   B is a binding agent capable of selectively binding to a nerve        cell surface receptor and mediating absorption of the compound        by the nerve cell;    -   IM is a non-cytotoxic imaging moiety which can be used to image        a nerve cell or an intracellular component of the nerve cell;        and    -   L is a linker coupling B to IM.

In regard to each of the above embodiments, particular classes ofbinding agents B which may be used include, but are not limited to,nucleic acid sequences, peptides, peptidomimetics, antibodies andantibody fragments. Examples of nucleic acids that can serve as thebinding agent B include, but are not limited to, DNA and RNA ligandsthat function as antagonists of nerve growth factors or inhibit bindingof other growth factors to nerve cell surface receptors. Examples ofpeptides that can serve as the binding agent B include, but are notlimited to, members of the nerve growth factors (neurotrophin) familysuch as NGF, BDNF, NT-3, NT-4, NT-6; derivatives, analogs, and fragmentsof nerve growth factors such as recombinant molecules of NGF and BDNF;and synthetic peptides that bind to nerve cell surface receptors andhave agonist or antagonist activities of nerve growth factors.

Antibodies, derivatives of antibodies and antibody fragments can alsoserve as the binding agent B. Examples of this type of binding agent Binclude, but are not limited to, anti-human trkA monoclonal antibody 5C3and anti-human p75 monoclonal antibody MC192.

The therapeutic moiety TM is selected to perform a non-cytotoxictherapeutic function within nerve cells. Examples of non-cytotoxicfunctions which the therapeutic moiety TM may perform include, but arenot limited to, the functions performed by adrenergic agents,analgesics, anti-trauma agents, anti-viral agents, gene therapy agents,and hormones (growth factors, interferons, etc.). Examples of classes oftherapeutic moieties include, but are not limited to, adrenergic agents(e.g., epinephrine, norepinephrine, dopamine, etenolol), analgesics(e.g., opioids, codeine, oxycodone), anti-trauma agents, anti-viralagents (e.g., acyclovir, gancyclovir, AZT, ddI, ddC, etc.), gene therapyagents (e.g., DNAs or RNAs which introduce a gene or replace a mutatedgene), steroids (e.g., cortisone, progesterone, estrogen), and hormones(e.g., growth factors, interferons).

The imaging moiety IM is a non-cytotoxic agent which can be used tolocate and optionally visualize a nerve cell or an internal component ofthe nerve cell which has absorbed the imaging moiety. Fluorescent dyesmay be used as an imaging moiety IM. Radioactive agents which arenon-cytotoxic may also be an imaging moiety IM.

In general, the linker may be any moiety which can be used to link thebinding agent B to the moiety M. In one particular embodiment, thelinker is a cleavable linker. The use of a cleavable linker enables themoiety M linked to the binding agent B to be released from the compoundonce absorbed by the nerve cell. The cleavable linker may be cleaved bya chemical agent, enzymatically, due to a pH change, or by being exposedto energy. Examples of forms of energy which may be used include light,microwave, ultrasound, and radiofrequency.

The present invention also relates to a method for selectivelydelivering a moiety into nerve cells comprising the steps of:

delivering to a patient a compound having the general formula:B-L-M

where:

-   -   B is a binding agent capable of selectively binding to a nerve        cell surface receptor and mediating absorption of the compound        by the nerve cell;    -   M is a moiety which performs a useful non-cytotoxic function        when absorbed by a nerve cell; and    -   L is a linker coupling B to M.

having the compound selectively bind to a nerve cell surface receptorvia the binding agent B; and

having the compound be absorbed by the nerve cell mediated by thebinding of the binding agent B to the nerve cell surface receptor.

In one embodiment, moiety M is a therapeutic moiety TM as describedherein and in another embodiment is an imaging moiety IM.

The above method can be used to deliver therapeutic moieties fortreating a variety of neurological disorders when the therapeutic moietyTM is a moiety useful for treating such neurological disorders.

The above method can be used to deliver therapeutic moieties fortreating pain when a therapeutic moiety TM for treating pain, such as ananalgesic, is included as the therapeutic moiety TM in the compound.

The above method can also be used to deliver steroid hormones fortreating nerve damage when a therapeutic moiety TM for treating nervedamage, such as a steroid hormone, is included as the therapeutic moietyTM in the compound.

The above method can also be used to stimulate nerve growth when atherapeutic moiety TM for inducing the production of a nerve growthfactor is included as the therapeutic moiety TM in the compound.

The above method can also be used to treat infected nerve cells infectedwith viruses or immunize nerve cells from viruses when the therapeuticmoiety TM in the compound is an antiviral agent.

The above method can also be used to perform gene thereapy when thetherapeutic moiety TM is a gene therapy agent.

DETAILED DESCRIPTION

The present invention relates to compounds which include a binding agentwhich binds to a nerve cell surface receptor and facilitates absorptionof the compound by the nerve cell; and a moiety. Different Moieties maybe included in the compounds of the present invention includingtherapeutic moieties that are non-cytotoxic to the nerve cells andimaging moieties which can be used to image nerve cells which absorbthese compounds.

In one embodiment, compounds of the present invention have the generalformula:B-L-TM

where:

-   -   B is a binding agent capable of selectively binding to a nerve        cell surface receptor and mediating absorption of the compound        by the nerve cell;    -   TM is a therapeutic moiety which has a non-cytotoxic therapeutic        effect when absorbed by a nerve cell; and    -   L is a linker coupling B to TM.

According to this embodiment, the binding agent B serves as a homingagent for nerve cells by selectively binding to nerve cell surfacereceptors. The binding agent B also serves to facilitate absorption ofthe compound by the nerve cell. The binding agent B can be any moleculewhich can perform these two functions. Particular classes of bindingagents which may be used include, but are not limited to, nucleic acidsequences, peptides, peptidomimetics, antibodies and antibody fragments.

Examples of nucleic acids that can serve as the binding agent B include,but are not limited to, DNA and RNA ligands that function as antagonistsof nerve growth factors or inhibit binding of other growth factors tonerve cell surface receptors (Binkley, J., et al., Nucleic Acid Res. 23:3198-3205 (1995); Jellinek, D., et al., Biochem. 33:10450-10456 (1994)).

Examples of peptides that can serve as the binding agent B include, butare not limited to, members of the nerve growth factors (neurotrophin)family such as NGF, BDNF, NT-3, NT-4, NT-6, etc. (see reviews: Frade, J.M., et al., Bioessays 20: 137-145 (1998); Shieh, P. B., Curr. Biol. 7:R627-R630 (1997); Dechant, G., et al., Curr. Opin. Neurobiol. 7: 413-418(1997); Chao, M. V. and Hempstead, B. L., Trends Neurobiol. 18: 321-326(1995)); and derivatives, analogs, and fragments of nerve growth factorssuch as recombinant molecules of NGF and BDNF (Ibanez et al., EMBO J.10: 2105-2110; Ibanez et al., EMBO J. 12: 2281-2293), synthetic peptidesthat bind to nerve cell surface receptors and have agonist or antagonistactivities of nerve growth factors (Longo, F. M., et al., CellRegulation 1: 189-195 (1990); LeSauteur, L. et al., J. Biol. Chem. 270:6564-6569 (1995); Longo F. M., et al., J. Neurosci. Res. 48: 1-17;Longo, et al., Nature Biotech. 14: 1120-1122 (1997)).

Examples of antibodies, derivatives of antibodies and antibody fragmentsthat can serve as the binding agent B include, but are not limited to,anti-human trkA monoclonal antibody 5C3 (Kramer, K., et al., Eur. J.Cancer 33: 2020-2091 (1997)), anti-human p75 monoclonal antibody MC192(Maliatchouk, S. and Saragovi, H. U., J. Neurosci. 17: 6031-7).

According to this embodiment, the therapeutic moiety TM is selected toperform a non-cytotoxic therapeutic function within nerve cells.Examples of non-cytotoxic functions which the therapeutic moiety TM mayperform include, but are not limited to, the functions performed byanalgesics, anti-trauma agents, anti-viral agents, gene therapy agents,and hormones (growth factors, interferons, etc.). Examples of classes oftherapeutic moieties include, but are not limited to, adrenergic agents(e.g., epinephrine, norepinephrine, dopamine, etenolol), analgesics(e.g., opioids, codeine, oxycodone), anti-trauma agents, anti-viralagents (e.g., acyclovir, gancyclovir, AZT, ddI, ddC, etc.), gene therapyagents (e.g., DNAs or RNAs which introduce a gene or replace a mutatedgene), steroids (e.g., cortisone, progesterone, estrogen), and hormones(e.g., growth factors, interferons).

The linker L serves to link the binding agent B to the therapeuticmoiety TM. A wide variety of linkers are known in the art for linkingtwo molecules together, particularly, for linking a moiety to a peptideor nucleic acid, all of which are included within the scope of thepresent invention.

Examples of classes of linkers that may be used to link the bindingagent B to the therapeutic moiety TM include amide, alkylamine, thiolether, alkyl, cycloalkyl, aryl linkages such as those described inHermanson, G. T., Bioconjugate Techniques (1996), Academic Press, SanDiego, Calif.

In certain applications, it is desirable to release the therapeuticmoiety TM once the compound has entered the nerve cell, resulting in arelease of the therapeutic moiety TM. Accordingly, in one variation, thelinker L is a cleavable linker. This enables the therapeutic moiety TMto be released from the compound once absorbed by the nerve cell. Thismay be desirable when the therapeutic moiety TM has a greatertherapeutic effect when separated from the binding agent. Thetherapeutic moiety TM may have a better ability to be absorbed by anintracellular component of the nerve cell when separated from thebinding agent. Accordingly, it may be necessary or desirable to separatethe therapeutic moiety TM from the compound so that the therapeuticmoiety TM can enter the intracellular compartment.

Cleavage of the linker releasing the therapeutic moiety may be as aresult of a change in conditions within the nerve cells as compared tooutside the nerve cells, for example, due to a change in pH within thenerve cell. Cleavage of the linker may occur due to the presence of anenzyme within the nerve cell which cleaves the linker once the compoundenters the nerve cell. Alternatively, cleavage of the linker may occurin response to energy or a chemical being applied to the nerve cell.Examples of types of energies that may be used to effect cleavage of thelinker include, but are not limited to light, ultrasound, microwave andradiofrequency energy.

The linker L used to link the binding agent B to the therapeutic moietyTM may be a photolabile linker. Examples of photolabile linkers includethose linkers described in U.S. Pat. No. 5,767,288 and U.S. Pat. No.4,469,774. The linker L used to link the binding agent B to thetherapeutic moiety TM may also be an acid labile linker. Examples ofacid labile linkers include linkers formed by using cis-aconitic acid,cis-carboxylic alkatriene, polymaleic anhydride, and other acidlabilelinkers, such as those linkers described in U.S. Pat. Nos. 5,563,250 and5,505,931.

Further examples of cleavable linkers include, but are not limited tothe linkers described in Lin, et al., J. Org. Chem. 56:6850-6856 (1991);Ph.D. Thesis of W.-C. Lin, U. C. Riverside, (1990); Hobart, et al., J.Immunological Methods 153: 93-98 (1992); Jayabaskaran, et al.,Preparative Biochemistry 17(2): 121-141 (1987); Mouton, et al., Archivesof Biochemistry and Biophysics 218: 101-108 (1982); Funkakoshi, et al.,J. of Chromatography 638:21-27 (1993); Gildea, et al., TetrahedronLetters 31: 7095-7098 (1990); WO 85/04674; and Dynabeads (Dynal, Inc., 5Delaware Drive, Lake Success, N.Y. 11042).

In another embodiment, compounds of the present invention have thegeneral formula:B-L-IM

where:

-   -   B is a binding agent capable of selectively binding to a nerve        cell surface receptor and mediating absorption of the compound        by the nerve cell;    -   IM is a non-cytotoxic imaging moiety which can be used to image        the nerve cell or an intracellular component of the nerve cell;        and    -   L is a linker coupling B to IM.

According to this embodiment, the binding agent B and linker L may bevaried as described above with regard to compounds having the generalformula B-L-TM. Further according to this embodiment, the imaging moietyIM may be a non-cytotoxic moiety which can be used to image nerve cells.Examples of imaging moieties that may be used include fluorescent dyesand radioisotopes which are non-cytotoxic.

The present invention also relates to a method for selectivelydelivering a non-cytotoxic therapeutic moiety into nerve cellscomprising the steps of:

delivering to a patient a therapeutic amount of a compound having thegeneral formula:B-L-TM

where:

-   -   B is a binding agent capable of selectively binding to a nerve        cell surface receptor and mediating absorption of the compound        by the nerve cell,    -   TM is a therapeutic moiety which has a non-cytotoxic therapeutic        effect when absorbed by a nerve cell, and    -   L is a linker coupling B to TM;

having the compound selectively bind to a nerve cell surface receptorvia the binding agent B; and

having the compound be absorbed by the nerve cell mediated by thebinding of the binding agent B to the nerve cell surface receptor.

The method of the present invention offers the advantage of specificallytargeting a non-cytotoxic therapeutic moiety to nerve cells where thetherapeutic moiety is absorbed by the nerve cells. The method utilizesthe fact that internalization of the conjugated agent is mediated by thebinding of the binding agent B to nerve cell surface receptors. Onceinternalized, the therapeutic moiety can accumulate within the nervecells where it has a therapeutic effect. The ability to selectivelydeliver the compound to nerve cells reduces the overall amount oftherapeutic moiety which needs to be administered. Selective delivery ofthe therapeutic moiety to the nerve cell reduces the amount of sideeffects observed due to non-specific administration of the therapeuticmoiety. In addition, the therapeutic moiety is less likely to beseparated from the binding agent and non-specifically administered ascompared to delivery methods involving the use of a binding agent and atherapeutic moiety in combination.

The method of the present invention can be used to deliver therapeuticmoieties for treating a variety of neurological disorders including, butnot limited to, Alzheimer's disease, Parkinson's disease, multiplesclerosis, neurodegenerative disease, epilepsy, seizure, migraine,trauma and pain. Examples of neuropharmaceuticals that may be usedinclude proteins, antibiotics, adrenergic agents, anticonvulsants,nucleotide analogs, anti-trauma agents, peptides and other classes ofagents used to treat or prevent a neurological disorders. For example,analgesics such as opioids, codeine and oxycodone can be conjugated tothe binding agent B and specifically delivered to the nerve cells. Sincethe same level of pain relief can be achieved using a smaller dosage ofanalgesics, side effects such as respiratory depression or potentialdrug addiction can be avoided or at least ameliorated. Steroid hormonessuch as corticosteriods can also be conjugated with nerve cell-specificbinding agents and used to treat inflammation of the nerves, which mayreduce the side effects associated with high doses of steroids, such asweight gain, redistribution of fat, increase in susceptibility toinfection, and avascular necrosis of bone.

The method according to the present invention can also be used todeliver agents that induce the production of nerve growth factor in thetarget nerve cells, especially under conditions of pathogenicunder-expression of NGFs (See Riaz, S. S. and Tomlinson, D. R. Prog.Neurobiol. 49: 125-143 (1996)). NGF induction has been demonstrated in awide variety of cell types, such as fibroblasts (Furukawa, Y. et al.,FEBS Lett. 247: 463-467(1989)), astrocytes (Furukawa, Y. et al., FEBSLett. 208: 258-262 (1986)), Schwann cells (Ohi, T. et al., Biochem. Int.20:739-746 (1990)) with a variety of agents including cytokines,steriods, vitamins, hormones, and unidentified components of serum.Specific examples of agents known to induce NGF include4-methylcatechol, clenbuterol, isoprenaline, L-tryptophan,1,25-dihydroxyvitamin D3, forskolin, fellutamide A, gangliosides andquinone derivatives (Riaz, S. S. and Tomlinson, D. R. Prog. Neurobiol.49: 125-143 (1996)).

The method according to the present invention can also be used todeliver antiviral drugs into nerve cells in order to treat diseasescaused by viral infection, to eliminate viruses spread to the nerves,and to inhibit infection by such viruses. Examples of viruses thatinfect the nervous system include but are not limited to rabies viruses,herpes viruses, polioviruses, arboviruses, reoviruses, pseudorabies,corona viruses, and Borna disease viruses. For example, antiviral drugssuch as acyclovir, gancyclovir, and Cifodovir can be conjugated to thebinding agent and used to inhibit active or latent herpes simplexviruses in the peripheral and central nervous system. Specific deliveryof the conjugate containing these antiviral drugs to the nervous systemcan reduce the side effects associated with high doses or long-termadministration of these drugs, such as headaches, rash and paresthesia.

The method according to the present invention can also be used todeliver marker compounds to image intracellular components of the nervecells. Such marker compounds include but are not limited to fluorescentdyes, radioactive complexes, and other luminophores.

The method according to the present invention can also be used toperform gene therapy wherein nucleic acids (DNA or RNA) are delivered tothe nerve cells. These nucleic acids may serve to replace genes whichare either defective, absent or otherwise not properly expressed by thepatient's nerve cell genome.

The above and other features and advantages of the present inventionwill become more apparent in the following description of the preferredembodiments in greater detail.

1. Binding Agent (B)

According to the present invention, a compound with a binding agent B isused to selectively deliver the conjugated therapeutic moieties TM tonerve cells. At the nerve cell, the binding agent B interacts with areceptor on the nerve cell and is absorbed by the nerve cell mediated bythis interaction. Any molecules possessing these two physical propertiesare intended to fall within the scope of a binding agent B as it is usedin the present invention. In particular, peptides or proteins with thesefeatures can serve as a binding agent B, examples including but notlimited to nerve growth factors (neurotrophins), antibodies againstnerve cell-specific surface proteins, mutants and synthetic peptidesderived from these peptides or proteins.

In one embodiment, neurotrophins are preferably used as the bindingagent B. Neurotrophins are a family of small, basic polypeptides thatare required for the growth, development and survival of neurons. Aparticular “survival” factor is taken up by the neuron via binding toone or more of a related family of transmembrane receptors. Table Ilists several members of the neurotrophin family and their cognatereceptors.

As listed in Table 1, nerve growth factor (NGF) is the first identifiedand probably the best characterized member of the neurotrophin family.It has prominent effects on developing sensory and sympathetic neuronsof the peripheral nervous system. Brain-derived neurotrophic factor(BDNF) has neurotrophic activities similar to NGF, and is expressedmainly in the CNS and has been detected in the heart, lung, skeletalmuscle and sciatic nerve in the periphery (Leibrock, J. et al., Nature,341:149-152 (1989)). Neurotrophin-3 (NT-3) is the third member of theNGF family and is expressed predominantly in a subset of pyramidal andgranular neurons of the hippocampus, and has been detected in thecerebellum, cerebral cortex and peripheral tissues such as liver andskeletal muscles (Ernfors, P. et al., Neuron 1: 983-996 (1990)).Neurotrophin-4 (also called NT-4/5) is the most variable member of theneurotrophin family. Neurotrophin-6 (NT-6) was found in teleost fish andbinds to p75 receptor.

As listed in Table 1 at least two classes of transmembrane glycoproteins(trk and p75) have been identified which serve as receptors forneurotrophins. The trk receptors (tyrosine kinase-containing receptor)bind to neurotrophins with high affinity, whereas the p75 receptorspossess lower affinity to neurotrophins. For example, nerve growthfactor (NGF) binds to a relatively small number of trkA receptors withhigh affinity (K_(D)=10⁻¹¹) and to more abundant p75 with lower affinity(K_(D)=10⁻⁹). The receptor-bound NGF is internalized with membrane-boundvesicles and retrogradely transported the neuronal cell body. Thus,native neurotropins may serve as the binding agent B in the compoundaccording the present invention to deliver the conjugated therapeuticagent TM to the neuronal cell body.

TABLE 1 The Neurotrophin Family and Its Receptors. Receptor Responsiveneurons Factor Kinase isoforms Nonkinase forms (examples) NGF trkA p75Cholinergic forebrain neurons Sympathetic ganglia DRG nociceptive BDNFtrkB p75^(LNTR) Many CNS populations trkB_(T1) Vestibular gangliatrkB_(T2) Nodose ganglia DRG mechanoreceptors NT-3 trkC p75^(LNTR) ManyCNS populations trkB and trkA Choclear ganglia NonpreferredtrkC_(TK-113) DRG proprioceptive trkC_(TK-108) NT-4 trk B p75 Many CNSpopulations trkB_(T1) Nodose ganglia trkB_(T2) Petrosalganglia NT-6 trkAp75

In addition to the neurotrophins described above, analogs andderivatives of neurotrophins may also serve as the binding agent B. Thestructure of mouse NGF has been solved by X-ray crystallography at 2.3 Aresolution (McDonald et al., Nature, 345: 411-414, (1991)). Murine NGFis a dimeric molecule, with 118 amino acids per protomer. The structureof the protomer consists of three antiparallel pairs of beta strandsthat form a flat surface, four loop regions containing many of thevariable residues between different NGF-related molecules, which maydetermine the different receptor specificities, and a cluster ofpositively charged side chains, which may provide a complementaryinteraction with the acidic low-affinity NGF receptor. Murine NGF has atertiary structure based on a cluster of three cysteine disulfides andtwo extended, but distorted beta-hairpins. One of these β-hairpin loopswas formed by the NGF 29-35 region. Structure/function relationshipstudies of NGF and NGF-related recombinant molecules demonstrated thatmutations in NGF region 25-36, along with other β-hairpin loop andnon-loop regions, significantly influenced NGF/NGF-receptor interactions(Ibanez et al., EMBO J., 10, 2105-2110, (1991)). Small peptides derivedfrom this region have been demonstrated to mimic NGF in binding to trkAreceptor and affecting biological responses (LeSauteur et al. J. Biol.Chem. 270, 6564-6569, 1995). Dimers of cyclized peptides correspondingto β-loop regions of NGF were found to act as partial NGF agonists inthat they had both survival-promoting and NGF-inhibiting activity whilemonomer and linear peptides were inactive (Longo et al., J. Neurosci.Res., 48, 1-17, 1997). Cyclic peptides have also been designed andsynthesized to mimic the β-loop regions of NGF, BDNF, NT3 and NT-4/5.Certain monomers, dimers or polymers of these cyclic peptides may have athree-dimensional structure which binds to neurotrophin receptors underphysiological conditions. All of these structural analogs ofneurotrophins that bind to nerve cell surface receptors and areinternalized can serve as the binding agent B of the compound accordingto the present invention to deliver the conjugated therapeutic moiety TMto the nervous system.

Alternatively, antibodies against nerve cell surface receptors that arecapable of binding to the receptors and being internalized can alsoserve as the binding agent B. For example, monoclonal antibody (MAb) 5C3is specific for the NGF docking site of the human p140 trkA receptor,with no cross-reactivity with human trkB receptor. MAb 5C3 and its Fabmimic the effects of NGF in vitro, and image human trk-A positive tumorsin vivo (Kramer et al., Eur. J. Cancer, 33, 2090-2091, (1997)).Molecular cloning, recombination, mutagenesis and modeling studies ofMab 5C3 variable region indicated that three or less of itscomplementarity determining regions (CDRS) are relevant for binding totrkA. Assays with recombinant CDRs and CDR-like synthetic polypeptidesdemonstrated that they had agonistic bioactivities similar to intact Mab5C3. Monoclonal antibody MC192 against p75 receptor has also beendemonstrated to have neurotrophic effects. Therefore, these antibodiesand their functionally equivalent fragments can also serve as thebinding agent B of the compound according to the present invention todeliver the conjugated therapeutic agent TM into the nerve cells.

Alternatively, peptidomimetics that are synthesized by incorporatingunnatural amino acids or other organic molecules may serve as thebinding agent B of the compound according to the present invention todeliver the conjugated therapeutic agent TM into the nerve cells. Thesesynthetic peptide mimics are capable of binding to the nerve cellsurface receptor and being internalized into the cell.

It is noted that the identification and selection of moieties which canserve as binding agents in the present invention can be readilyperformed by attaching an imaging moiety IM to the potential bindingagent in order to detect whether the potential binding agent isinternalized by the nerve cells. In this regard, combinatorial andmutagenesis approaches may be used to identify analogs, derivatives andfragments of known binding moieties which may also be used as bindingmoieties according to the present invention.

2. Therapeutic Moiety (TM)

An aspect of the present invention relates to the delivery of compoundsinto nerve cells which are non-cytotoxic to the nerve cells and performa therapeutic function. Examples of therapeutic functions include, butare not limited to, treatment of neurological disorders, gene therapy,intracellular target imaging, cell sorting, or separation schemes.Examples of classes of therapeutic moieties include, but are not limitedto adrenergic agents such as epinephrine, norepinephrine, dopamine,etenolol; analgesics such as opioids, codeine, oxycodone; anti-traumaagents; anti-viral agents such as acyclovir, gancyclovir, AZT, ddI, ddC;gene therapy agents such as; steroids such as cortisone, progesterone,estrogen; and hormones such as growth factors and interferons. Suchcompounds may optionally also include an imaging moiety, such asfluorescent moieties, for imaging intracellular components of the nervecells.

3. Linker (L)

According to the present invention, a binding agent B is linked to atherapeutic moiety TM by a linker L. In general, any method of linking abinding agent to a therapeutic moiety may be used and is intended tofall within the scope of the present invention.

Many different types of linkers have been developed for cross linkingproteins and conjugating proteins or peptides with other agents. Theselinkers include zero-length cross linkers, homobifunctionalcross-linkers, heterobifunctional cross-linkers and trifunctionalcross-linkers. These linkers may have different susceptibility tocleavage under certain conditions. Depending on a particular applicationaccording to the present invention, an appropriate linker may be chosen.When an intracellular release of the agent from its conjugate isdesired, a cleavable linker is chosen which is susceptible to cleavageby external stimuli such as light and heat, by intracellular enzymes, orby a particular microenvironment inside the cell.

In one embodiment, the linker L has one of the following generalstructures:B—R₁—(CO)—NH—R₂-TMB—R₃—NH—R₄-TMB—R₃—S—R₄-TMB—R₅—(CH₂)_(n)—R₆-TM

Wherein R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from thegroup consisting of alkyls, aryls, heteroaryls, cycloalkyls,cycloalkenes and heterocycloalkenes.

4. Cleavable Linkers

One particular embodiment of the present invention relates to compoundswhich include a cleavable linker L. In some instances, the therapeuticmoiety TM is more efficacious or potent when free from a carriermolecule such as a binding agent. In such instances, it is desirable toutilize a cleavable linker which allows the therapeutic moiety TM to bereleased from the compound once inside the cell.

Many cleavable linker groups have been developed which are susceptibleto cleavage and by a wide variety of mechanisms. For example, linkershave been developed which may be cleaved by reduction of a disulfidebond, by irradiation of a photolabile bond, by hydrolysis of derivatizedamino acid side chain, by serum complement-mediated hydrolysis, and byacid-catalyzed hydrolysis.

Examples of photolabile linkers that may be used include those linkersdescribed in U.S. Pat. No. 5,767,288 and U.S. Pat. No. 4,469,774.

Acid-labile linkers are preferred in the practice of the presentinvention by taking advantage of a cell's receptor-mediated endocytosispathways. Receptors that are internalized by receptor-mediatedendocytosis pass through acidified compartments known as endosomes orreceptosomes. Since the interior of the endosomal compartment is keptacidic (pH˜6.0) by ATP-driven H⁺ pumps in the endosomal membrane thatpump H⁺ into the lumen from the cytosol, a change in pH within the nervecell can be used to cause the acid-labile linker to be cleaved andrelease the therapeutic moiety. Examples of acid labile linkers whichmay be used include the cis-aconitic acid, cis-carboxylic alkatriene,polymaleic anhydride, and other acid labile linkers described in U.S.Pat. Nos. 5,563,250 and 5,505,931.

5. Examples of Compounds According to the Present Invention

Table 2 provides several compounds according to the present invention.It is noted that in each instance, the particular therapeutic moieties,binding moieties, and linkers shown may be interchanged with othersuitable therapeutic moieties, binding moieties, and linkers. In thisregard, the compounds shown in the table are intended to illustrate thediversity of compounds provided according to the present invention.

TABLE 2

wherein B is selected from the group consisting of nerve growth factorsNGF, BDNF, NT-3, NT-4, NT-6, anti-neurotrophin receptor antibodies MAb5C3 and Mab MC192.

wherein B is selected from the group consisting of nerve growth factorsNGF, BDNF, NT-3, NT-4, NT-6, anti-neurotrophin receptor antibodies MAb5C3 and Mab MC192.

wherein B is selected from the group consisting of nerve growth factorsNGF, BDNF, NT-3, NT-4, NT-6, anti-neurotrophin receptor antibodies MAb5C3 and Mab MC192.

wherein B is selected from the group consisting of nerve growth factorsNGF, BDNF, NT-3, NT-4, NT-6, anti-neurotrophin receptor antibodies MAb5C3 and Mab MC192.

wherein B is selected from the group consisting of nerve growth factorsNGF, BDNF, NT-3, NT-4, NT-6, anti-neurotrophin receptor antibodies MAb5C3 and Mab MC192.6. Methods for Using Compounds of the Present Invention

Described below are several methods for formulating and administeringthe compounds of the present invention. The compounds of the presentinvention may be employed in these and other applications.

a. Pharmaceutical Formulations Utilizing Compositions of the PresentInvention

The compounds of the present invention may be incorporated into avariety of pharmaceutical compositions including, but not limited to: asterile injectable solution or suspension; hard or soft gelatincapsules; tablets; emulsions; aqueous suspensions, dispersions, andsolutions; suppositories. Other pharmaceutically suitable formulationsfor delivering the compounds of the present invention to nerve cells mayalso be used and are intended to fall within the scope of the presentinvention.

b. Routes of Administration

The compounds according to the present invention can be administeredorally, by subcutaneous or other injection, intravenously,intracerebrally, intramuscularly, parenternally, transdermally, nasallyor rectally. The form in which the compound is administered depends atleast in part on the route by which the compound is administered.

While the present invention is disclosed with reference to preferredembodiments and examples detailed above, it is to be understood thatthese examples are intended in an illustrative rather than limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, which modifications will be within the spiritof the invention and the scope of the appended claims. The patents,papers, and books cited in this application are to be incorporatedherein in their entirety.

1. A composition comprising: a compound having the general formula:B-L-TM where: B is nerve growth factor (NGF) or a fragment thereof whichselectively binds to a neurotrophin receptor; TM is a noncytotoxic,antiviral moiety other than the nerve growth factor or fragment thereof;and L is a cleavable linker coupling B to TM; and a pharmaceuticallyacceptable vehicle.
 2. The composition of claim 1, wherein B is an NGFfragment capable of binding to TrkA and triggering absorption of thecompound.
 3. The composition of claim 1, wherein B is an NGF fragmentcapable of binding to p75 and triggering absorption of the compound. 4.The composition of claim 1, wherein said antiviral moiety is acyclovir.5. The composition of claim 1, wherein said antiviral moiety isganciclovir.
 6. The composition of claim 1, wherein said antiviralmoiety is AZT.
 7. The composition of claim 1, wherein said antiviralmoiety is cidofovir.
 8. The composition of claim 1, wherein saidantiviral moiety is ddI.
 9. The composition of claim 1, wherein saidantiviral moiety is ddC.
 10. The composition of claim 1, wherein saidcleavable linker is an amide linker.
 11. The composition of claim 1,wherein said cleavable linker is an alkylamine linker.
 12. Thecomposition of claim 1, wherein said cleavable linker is a thioetherlinker.
 13. The composition of claim 1, wherein cleavable linker is analkyl linker.
 14. The composition of claim 1, wherein said cleavablelinker is a cycloalkyl linker.
 15. The composition of claim 1, whereinsaid cleavable linker is an aryl linker.